Proper control of mitosis is critical to maintain the stability of the genome during cell proliferation, and genomic instability may contribute directly to the generation of cancer. Study of mechanisms that regulate mitosis, therefore, is critical to understand how cancer develops, and to discover new ways to prevent and treat the disease. Mitosis is also an important target for cancer therapy. Recently, new selective anti-mitotic drugs such as aurora kinase inhibitors have shown great promise in pre-clinical experiments, and there is now immense interest in identifying new drug targets in mitosis. We have recently discovered a novel mitotic histone kinase, haspin, that has homologs in diverse eukaryotes. Human haspin mRNA is expressed in proliferating but not non-proliferating cells. During mitosis, haspin associates with condensed chromosomes, particularly at centromeres, and is responsible for phosphorylation of Thr-3 in histone H3. Haspin is also found at mitotic centrosomes. Importantly, haspin RNA interference causes misalignment of metaphase chromosomes and spindle defects, preventing completion of normal mitosis. These studies add haspin to the select group of kinases that regulate mitotic chromosome dynamics and spindle activity and provide the first indication that haspin, like the aurora kinases, might be a suitable target for cancer therapy. Further study of haspin action in mitosis and validation of haspin as a cancer drug target are currently limited, however, by the lack of specific small molecule inhibitors of the kinase. To identify small molecule inhibitors of haspin, we will develop an in vitro haspin kinase assay suitable for high-throughput screening of chemical libraries. In Aim 1 we will produce, in E. coli or the baculovirus system, functional full-length recombinant haspin for use in screening assays. In Aim 2 we will develop and optimize a homogenous time-resolved fluorescence kinase assay for haspin. An alternative strategy using a separation-based approach is also described. In Aim 3, we outline assays to confirm hits from the screening process and develop secondary screens to assess the inhibitory properties and functional effects of these compounds in vitro and in cells. Haspin inhibitors will provide a new approach to investigate the basic biology of cell division and will yield insights that cannot be obtained using existing technology. Furthermore, such inhibitors will provide an excellent way to validate haspin as a target for cancer treatment, and they might find direct application as chemotherapeutic drugs. [unreadable] [unreadable] [unreadable]